DISSERTATION ON
STUDY OF VITAMIN - D LEVELS IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE PATIENTS & HEALTHY VOLUNTEERS
Submitted to
THE TAMILNADU Dr. M. G. R. MEDICAL UNIVERSITY Chennai – 600032
In partial fulfilment of the requirement for the award of degree of
DOCTOR OF MEDICINE IN BIOCHEMISTRY BRANCH –XIII
Submitted by
Register number: 201723751
KARPAGA VINAYAGA INSTITUTE OF MEDICAL SCIENCES AND RESEARCH CENTRE
MADHURANTHAGAM TAMILNADU MAY 2020
CERTIFICATION
This is to certify that “Study of Vitamin D Levels in Chronic Obstructive Pulmonary Disease Patients & Healthy Volunteers” is a bonafide work of Dr.G.Gurulakshmi, in partial fulfilment of the requirements for the M.D
Biochemistry (Branch XIII) examination of The Tamilnadu Dr. M.G.R Medical University to be held on May 2020.
Dr. V.Santhosh., MD Dr. Sufala Sunil Viswas Rao., MD Professor and Guide Principal,
Head of Department, Karpaga Vinayaga Institute Department of Biochemistry of Medical Sciences,
Karpaga Vinayaga Institute Madhuranthagam.
of Medical Sciences, Madhuranthagam.
CERTIFICATION
This is to certify that “Study Of Vitamin- D Levels In Chronic Obstructive Pulmonary Disease Patients & Healthy Volunteers” is a bonafide work of Dr.G.Gurulakshmi, in partial fulfilment of the requirements for the M.D
Biochemistry (Branch XIII) examination of The Tamilnadu Dr.M.G.R Medical University to be held on May 2020.
Dr.V.SANTHOSH.,MD
Guide, Professor and Head Of Department, Department of Biochemistry,
Karpaga Vinayaga Institute of Medical Sciences, Madhuranthagam,
Kanchipuram Dt.
DECLARATION
I, Dr. GURULAKSHMI.G hereby declare that this dissertation “Study Of Vitamin- D Levels In Chronic Obstructive Pulmonary Disease Patients &
Healthy Volunteers” is a presentation of my own work and that it has not been submitted anywhere for any award.
Wherever contributions of others are involved, every effort is made to indicate this clearly, with due reference to literature and discussions.
This work was done under the guidance of Professor Dr.V.SANTHOSH., MD, at Karpaga Vinayaga Institute of Medical Sciences, Madhuranthagam.
Candidate’s Name: DR. GURULAKSHMI.G
Candidate’s signature:
Date:
In the capacity as guide for the candidate’s dissertation work, I certified that the above statements are true to the best of my knowledge.
Dr.V.SANTHOSH. MD,
Guide, Professor and Head of Department, Department of Biochemistry,
Karpaga Vinayaga Institute of Medical Sciences, Madhuranthagam,
Kanchipuram Dt.
ACKNOWLEDGEMENT
“If four things are followed – having a great aim, acquiring knowledge, hard work, and perseverance – then anything can be achieved”. – Dr. A.P.J. Abdul Kalam.
I bow myself in front of the Almighty and express my humble thankfulness for all his blessings throughout my life.
I express my thankfulness extreme lovingly to my Parents Mr.P.Gurusamy and Mrs.G.Sankareshwari for their blessings, care and for torching me a decisive path in my life.
I express my sincere and heartfelt gratitude to our respected Managing Director, Professor Dr.R.Annamalai., M.S., for permitting and for extending his valuable support in conducting this study.
I humbly respect and thank our Medical Director Dr.Sathiyanarayanan Srinivasan. DA, and Principal Dr. Sufala Sunil Viswas Rao., M.D., for permitting and supporting me to conduct this study.
I acknowledge my deep sense of gratitude and regards to my Professor and guide Dr. V.SANTHOSH. MD, Head of department of Biochemistry, KIMS &
RC, Maduranthagam, for his guidance, valuable suggestions, and endless encouragement, which he lovingly bestowed upon me, in the preparation of this dissertation. His Constant scrutiny, during sample collection and processing, suggestions in writing dissertation was the immense help in completing the study for which I shall remain forever indebted to him.
I would like to thank my former Head of Department of Biochemistry, Professor Dr.Aruna Kumari. R. MD, for her guidance in choosing this topic. She does not put undue pressure. She is the one who taught the virtue of simplification and understanding in learning. She always greets us with a beautiful smile and shows that Knowledge is her power.
I would like to express my sincere gratitude to Dr.A.Khadeja Bi MD, Associate Professor for her unflinching support and encouragement in her busy schedule throughout this study.
I would like to thank Dr. L. Siva Msc., phD., Associate Professor for his kind support for my study.
I extend my immense thanks and gratitude to Dr. Murugesan DTCD., Senior resident, Department of Pulmonary medicine, KIMS & RC for his tremendous support and boundless guidance in referring cases for sample collection.
I thank my department faculties Dr.Amar Nakeshkumar., Ph.D., Mr.Saravanan.,M.Sc.,Ph.D., Mrs.S.Suganya.,M.Sc., Dr.M.Gomathi, MD for their support during the course of the study.
I also wish to thank Non-Medical Demonstrator, our Laboratory Technicians of Undergraduate Laboratory and Central Research Laboratory for their help and Co-operation.
I would like to thank Miss.Adithya for her guidance towards statistical analysis techniques.
Many thanks to all the patients and volunteers who participated in the study.
I am grateful to my dear husband Dr.A.Senthilkumar., DNB, (Pulmonary medicine) who is a constant inspire for choosing this course and suggested this topic for my study. Without his unbounded, unconditional love, trust and support it would not be possible to complete this study.
Last but not least, I would like to thank my dear Son S.G. Keeran Kumar, a precious gift to my life. He taught me the patience and capability to manage difficult situations by his daily smiles. He would have been a right choice for whom my dissertation is dedicated too.
PLAGIARISM
TABLE OF CONTENTS
S.NO. TITLE PAGE
NO.
1. INTRODUCTION 1-3
2. AIMS AND OBJECTIVES 4
3. REVIEW OF LITERATURE 5-38
4. MATERIALS AND METHODS 39-56
5. RESULTS 57-73
6. DISCUSSION 74-76
7. CONCLUSION 77-78
8. BIBLIOGRAPHY 79-90
9. ANNEXURES 91-98
10. MASTER CHART 99-102
ABBREVIATIONS LIST OF ABBREVIATIONS ABEI N-aminobutyrl-N-ethylisoluminol
BP BLOOD PRESSURE
BTS BRITISH THORACIC SOCIETY
BOLD BURDEN OF OBSTRUCTIVE LUNG DISEASES COPD CHRONIC OBSTRUCTIVE PULMONARY DISEASE
CRD CHRONIC RESPIRATORY DISEASE DALY DISABILITY ADJUSTED LIFE YEARS
FITC FLUORESCEIN ISOTHIOCYANATE FEV FORCED EXPIRATORY VOLUME
FEV1 FORCED EXPIRATORY VOLUME IN ONE SECOND FRC FUNCTIONAL RESIDUAL CAPACITY
FVC FORCED VITAL CAPACITY
GOLD GLOBAL INITIATIVE FOR CHRONIC OBSTRUCTIVE LUNG DISEASE
GLDH GLUTAMATE DEHYDROGENASE
Hb HEMOGLOBIN
HPC HEMATOPOEITIC PROGENITOR CELL INSEARCH
INDIAN STUDY ON EPIDEMIOLOGY OF ASTHMA,
RESPIRATORY SYMPTOMS AND CHRONIC BRONCHITIS IN ADULTS
MHFW MINISTRY OF HEALTH & FAMILY WELFARE MDI METERED DOSE INHALER
RLU RELATIVE LIGHT UNIT
RV RESIDUAL VOLUME
RPM REVOLUTION PER MINUTE TLC TOTAL LUNG CAPACITY WBC WHITE BLOOD CORPUSCLES
WHO WORLD HEALTH ORGANISATION
VC VITAL CAPACITY
1
INTRODUCTION:
“Chronic obstructive pulmonary disease (COPD)” is one of the most important cause for morbidity and mortality across the globe. According to the World Health Organization report nearly 65 million people are suffering moderate to severe COPD and it will be the third cause of death by 2020. [1] A newer projection estimated COPD will be the fourth leading cause of death in 2030.[2] In India prevalence of COPD is 5% in men & 2.7% in women.[3] About half a million people in India die due to COPD, which is four times the number of COPD deaths in Europe & USA.
Global Initiative for Chronic Obstructive Lung Disease (GOLD) program defines that “Chronic Obstructive Pulmonary Disease (COPD), is a common, preventable and treatable disease, is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. Exacerbations and co-morbidities contribute to the overall severity in individual patients.”[4]
Smoking is the major risk factor for COPD, however other factors like environmental tobacco smoking, occupational dust/smoke exposures, ambient air pollution, indoor air pollution, poorly treated asthma, previously treated tuberculosis, Malnutrition, and other environmental factors also contribute to COPD.
COPD is suspected in any patients aged above 40 years with chest symptoms like breathlessness, wheeze, cough, expectoration, chest pain along with strong exposure to risk factors. Very rarely the COPD do occur in younger patients like congenital Emphysema, α1 anti-trypsin deficiency syndrome.
Vitamin D deficiency is associated with pulmonary function deterioration.
2
Vitamin D deficiency is common across various populations as well as among several skeletal and nonskeletal conditions including autoimmune diseases, diabetes, pulmonary diseases. The patients with pulmonary diseases such as asthma and chronic obstructive pulmonary disease (COPD) are at greater risk of vitamin D deficiency.
The clinical manifestations of COPD are predominantly pulmonary symptoms like cough, expectoration, breathlessness and wheeze. Although the extra pulmonary manifestations like osteoporosis, dyslipidemia, hypertension, muscle weakness, psychosis, clubbing also occur commonly.
Osteoporosis is one of the important clinical manifestations of COPD patients which adversely affect the quality of life in COPD patients. Many patients with COPD confine themselves at home which is not only due to breathlessness or wheeze, but also due to severe bony pain, muscle wasting and generalized weakness. The skeletal occurs due to vitamin D deficiency following poor intake, limitation of physical activities and prolonged use of corticosteroids.
Many recent studies revealed that Vitamin D plays an key role in various diseases like COPD, cancer, cardiovascular disease, autoimmune disease, systemic hypertension, diabetes mellitus etc. [5,6,7] Among COPD patients Vitamin D deficiency do occur frequently in severe and very severe COPD patients and in frequent exacerbations.
Recent studies have showed evidence of vitamin D in protection against risk for cardiovascular diseases, malignant neoplasms and diabetes along with osteoporosis and other bone disorders. More than 2000 genes in the human genome respond to vitamin D. [102]
3
Many studies revealed that Vitamin D deficiency are common in patients with COPD and its prevalence among COPD varies from 31-77%.[8,9,10] The association of hypovitaminosis D with the prevalence, severity of COPD and its exacerbations has been carried out by a number of researches but there is a variations in the results. So, keeping this in view, this study was taken up with the following aims and objectives.
4
AIM AND OBJECTIVES:
1. To compare vitamin- D levels in COPD patients & healthy volunteers.
2. To analyse the level of vitamin-D levels in stable COPD pateints & in COPD patients with exacerbations.
3. To analyse the vitamin-D levels according the severity of COPD in patients.
5
REVIEW OF LITERATURE
Chronic Obstructive Pulmonary Disease is a brief term that includes chronic bronchitis and emphysema. [11] Chronic bronchitis is defined as “the presence of a chronic productive cough on most days for three months, in each of two consecutive years”. [11] Emphysema is the pathological term which is defined as abnormal, permanent enlargement of the distal air spaces, distal to the terminal bronchioles, accompanied by destruction of their walls and without obvious fibrosis.[11]
The description of emphysema was listed since the earlier medical era. Bonet’s described the term of “voluminous lungs” in 1679 (Bonet 1679); Morgagni described about 19 cases in 1769 in which the lungs were “turgid”, particularly from air; and Baille’s illustrations of the emphysematous lung, is thought to be that of Samuel Johnson (Baille 1789; Bishop 1959).[12]
Badham (1814) used the word ‘catarrh’ to refer to the chronic cough and mucus hyper secretion that are cardinal symptoms of chronic bronchitis. He described that bronchiolitis and chronic bronchitis are disabling disorders of the lungs (Badham 1814).[12]
Laennec a clinician, pathologist, and inventor of the stethoscope used the term the emphysema in 1821 in his Treatise of diseases of the chest after careful dissections of the patients that he had studied during life. He illustrated that emphysema was hyperinflation of lungs which did not empty well (Laennec 1821).[12]
Subsequently there are increased interest in COPD resulting in several
“hypothesis” for explaining the pathogenesis of airflow limitation.
6
The “Dutch hypothesis” suggested that asthma and airway hyperactivity could lead to fixed airflow limitation. [13] Whereas the “British hypothesis” stated the concept of mucus hyper secretion led to airway remodelling and airflow limitation. [14]
Laurell and Eriksson from Sweden described that protease- induced emphysema in animal models which led to concept of “protease- antiprotease hypothesis or Swedish hypothesis”.[15]
In addition an American pathologist Averill Liebow suggested that deficiency in maintaining lung structure, particularly of alveolar capillaries, could lead to emphysema which has been termed as “American hypothesis”. [16,17] Current evidence suggests that the mechanisms proposed by all these “hypothesis” contribute for developing COPD.
John Hutchinson invented the spirometer in 1846 (Hutchinson 1846) which is the key to the diagnosis and management of COPD. However the use of spirometry in diagnosis and management of COPD is still poorly applied in most of the locations in world today. Instrument made by Hutchinson’s only measured vital capacity. It took another 100 years to add the concept of timed vital capacity as a measure of airflow, for spirometry by Tiffeneau (Tiffeneau and Pinelli, 1947). [12]
In 1950,1951 Gaensler introduced the concept of the air velocity index on Tiffeneau’s work and later the forced vital capacity, which is the foundation of FEV1 and FEV1/FVC percent (Gaensler, 1950,1951).[12]
7
COPD in India
There were a few studies from India on prevalence of COPD before 20th Century.
Most of the studies were limited by small sample size and were based on unvalidated questionnaires.
In India, chronic respiratory diseases (CRDs) account for 3% of DALYs, and COPD is the major cause among CRDs.[18] COPD causes about 500,000 deaths per year.[19] A review of data from multiple sources suggested that COPD causes more deaths than tuberculosis and pneumonia. [20] Recently, the State Health Systems Resource Centre reported that COPD is the leading cause of death & its surpassing coronary artery disease, cerebrovascular accident and diabetes in Maharashtra state.[21]
Jindal et al showed that the prevalence of COPD among rural population of North India is 6.2% in males and 3.9% in females. He also showed that COPD prevalence is 4.2 % in males and 1.6% in females in the urban population of North India.[22]
Goel S et al, in a cross sectional study conducted in rural and urban areas in
shimla revealed that prevalence of COPD is 11.1% among males and 6.1% among females. [23]
Jindal SK et alin his study on prevalence of COPD showed that 4-10% of the adult population in India suffer from COPD.In addition regional COPD working group for 12 Asian Pacific Countries estimated prevalence of COPD to be 6.3%.[24].
The INSEARCH I and II (Indian Study on Epidemiology of Asthma, Respiratory Symptoms and Chronic Bronchitis in Adults) involved 121,776 individuals of more than 35 years of age, including both genders living in rural and urban population
8
over 16 centres in the country based on a well validated questionnaire. [25,26] According to these studies the prevalence of COPD in India was 3.7% (4.5% and 2.9% among males and females, respectively). The estimated burden of COPD in India is about 15 million cases (males and females contributing to 9.1 and 5.8 million, respectively).
INSEARCH phase II revealed that 60% of the chronic bronchitis patients were smokers.[27]
According to the preliminary report of the “Million Death Study”, Among Indian adults CRDs were the one of the second & third most common cause of death in rural and urban population, respectively. [28]
COPD poses enormous burden in terms of morbidity and mortality globally and also in India. The calculated economic loss in India due to COPD is about Rs.35,000 crores for the year 2011 and is assumed to exceed Rs.48,000 crores for the year 2016.[29]
These economic losses are more than the annual budget of the Ministry of Health and Family Welfare (MHFW) for the year 2010-2011, which was Rs.25,124 crores. [19]
In a study that assessed the costs of treatment amongst 423 COPD patients in India, was found that patients spents 15% of their annual income on smoking products and 30% on disease management. [30] It has been calculated that proper programme based or guideline-based management of COPD can reduce these costs by approximately 70%.[18]
9
Definitions:
American Thoracic Society defined COPD as “a disease state characterized by the presence of airflow obstruction due to chronic bronchitis or emphysema; the airflow obstruction is generally progressive, may be accompanied by airway reactivity and partially reversible.” [31]
European Respiratory Society has adopted a similar definition: “a disorder characterized by reduced maximum expiratory flow and slow forced emptying of the lungs; features which do not change markedly over several months. Most of the airflow limitation is slowly progressive and irreversible”. [32]
The definition adopted by the BTS (British Thoracic Society) is similar: “a slowly progressive disorder characterized by airways obstruction (reduced FEV1 and FEV1/VC ratio), which does not change markedly over several months”. Most of the lung function impairment is fixed, although some reversibility can be produced by bronchodilator (or other) therapy. [33]
Global Initiative for Chronic Obstructive Lung Disease (GOLD) program defines that “Chronic Obstructive Pulmonary Disease (COPD), is a common, preventable and treatable disease, is characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response in the airways and the lung to noxious particles or gases. Exacerbations and co-morbidities contribute to the overall severity in individual patients.” [100]
Joint ICS/NCCP (1) from India recommended the definition of COPD as
“Chronic Obstructive Pulmonary Disease (COPD) is a common, preventable lung
10
disorder characterized by progressive, poorly reversible airflow limitation often with systemic manifestations, in response to tobacco smoke and/ or other harmful inhalational exposures”.[34]
An exacerbation of COPD is defined as an event in the course of the disease characterized by a baseline dyspnoea, cough, and/or sputum that is beyond normal day-to-day variations. It is acute in onset, and may warrant a change in regular medication in a patient with underlying COPD.It is one of the important cause of ICU admissions, deterioration of pulmonary functional capacity of a COPD subject causing detoriation and death. [35]
Risk Factors:- [4]
Table 1: Showing risk factors for COPD
Established Probable
Tobacco smoking Outdoor air pollution Environmental tobacco smoke Pulmonary tuberculosis Exposure to biomass fuel smoke Poorly treated asthma
Occupational exposure Intrauterine growth retardation Alpha–1–antitrypsin deficiency Poor nourishment
Repeated lower respiratory tract infections during childhood
Low socio-economic status
11
PATHOLOGY [36, 37, 38]
Pathological changes of COPD are found both in airways, lung parenchyma, and pulmonary vessels. The trachea, bronchi, and bronchioles constitute the central airway, which is greater than 2-4 mm in internal diameter. The inflammatory cells like Neutrophils, Macrophages, T Lymphocytes, B Lymphocytes, Eosinophils and Epithelial cells infiltrate the surface epithelium leading to destruction of the lung parenchyma in COPD patients typically occurs as centrilobular emphysema. It involves dilatation and destruction of the respiratory bronchioles of the lung.
In COPD, Pulmonary vascular changes are initially characterized by a thickening of the intimal layer of the vessel wall. A greater amount of smooth muscle, proteoglycans, and collagens thicken the vessel wall when COPD worsens.
Endogenous cell damage and dysfunction produced by effects of cigarette smoke products or other inflammatory elements is considered to be the primary alteration that initiates sequence of events resulting in pulmonary hypertension. [36]
PATHOPHYSIOLOGY [39, 37, 40]
In the lung it leads to corresponding physiological changes, includes increase in mucus secretion, abnormal ciliary function, airflow limitation, pulmonary hyperinflation, abnormal in gas exchange, pulmonary hypertension and cor pulmonale, usually develops in this order.
Increased Mucus secrertion and abnormal ciliary function leads to chronic cough and sputum production. These symptoms can be present for many years. Other symptoms or physiological abnormalities develop later.
12
Spirometry is used to measure expiratory airflow limitation, which is the hallmark physiological change of COPD. It is a key to diagnosis of the disease. It is primarily due to fixed airways obstruction as well as increase in airways resistance.
The ability of the small airways to maintain patency is inhibited by decrease of alveolar attachment, plays a minor role.
In advanced COPD, peripheral airway obstruction, destruction of the parenchyma and pulmonary vascular abnormalities reduce the gas exchange, producing hypoxemia and hypercapnea.
Later, pulmonary hypertension develops, which is the major cardiovascular complication of COPD. It is associated with corpulmonale and a poor prognosis.
Patients with COPD have divided on clinical grounds and blood gas abnormalities into two extreme presentations. Type A patients or “pink puffers” have severe dyspnea, normal or increase paCO2, only a mild decrease in paO2 at rest, and low DLco. These patients are hypoxemic only at later stage in the disease and therefore do not have pulmonary hypertension, corpulmonale and consequently fluid retention and secondary polycythemia.
In contrast, “blue bloaters” or Type B patients present with cough and sputum production and are likely to develop hypoxemia and hypercapnea earlier in the disease course and hence corpulmonale, fluid retention and polycythemia. The pink puffers were thought to have predominant emphysema and the blue bloaters were the bronchitic type. Blue bloaters have almost two times increase in the mortality rate of pink puffers with similar degree of obstruction. [39]
13
CLINICAL FEATURES AND DIAGNOSIS [4, 41-46]
The common type of presentation of chronic obstructive pulmonary disease is a period of totally asymptomatic phase.
Symptoms:
Symptoms commonly associated with COPD includes dyspnea, cough with or without expectoration and wheezing. The severity of dyspnea is reported by patients seems parallel to severity of lung function. [41] The threshold for discernible exercise limitation occurs with forced expiratory volume (FEV1) value about 1.5 litres.
Dyspnea is usually gradual in onset and is present for many years. Chronic productive cough is frequently associated with dyspnea.
Hallmark of chronic bronchitis is cough with sputum production which develops suddenly, occurring first only in the morning and volume rarely exceeds 60mL of mucoid sputum. During exacerbation there is an excessive cough, purulent sputum, wheezing, dyspnea and intermittent fever which are occasional with mild degree. As the disease gets progressive, the period between the acute attacks become shorter.
As the disease progresses from chronic bronchitis to emphysema, dyspnoea increases and there is scanty sputum production. In this stage patients develop physical stigma of COPD like muscle wasting, weight loss, weakness, and fatigue. At this stage any exacerbation gives rise to erythrocytosis and patient develop polycythemia.
14
Retention of carbon dioxide (hypercapnea) which usually presents with morning headache and drowsiness due to lack of sleep at night. Patients with COPD tend to develop sleep apnoea and are associated with sudden decrease in saturation of oxygen, giving rise to sudden death. As the disease progresses patients develop corpumonale with right heart failure and edema.
Diagnosis:
Spirometry [47, 48]
Spirometry is an important tool for the diagnosis of COPD, as blood pressure measurements are for the diagnosis of hypertension. Spirometry is the most strong and healthy test of airflow limitation in patients with COPD. The diagnostic criteria for COPD are low FEV1 with an FEV1/FVC ratio below the normal range. The rate of decrease of FEV1, can be used to assess susceptibility, progression of disease and reversibility of the airflow obstruction in cigarette smokers.
Spirometric parameters used for diagnosis of COPD include
• FEV1
• FVC
• FEV1/FVC ratio
FEV1 is influenced by the age, sex, height, and ethnicity and is best considered as a percentage of the predicted normal value. The degree of abnormality in spirometry reflects the severity of COPD.
15
The ratio of FEV1/FVC is between 70% and 80% in normal adults; a value less than 70% indicates airflow limitation and the possibility of COPD. The severity of COPD is divided into mild, moderate, severe and very severe based on the FEV1% and presence of right heart failure. [4]
Three measurements are commonly made from a recording of forced exhaled volumes versus time, i.e. spirogram.
1) FEV1 (forced expiratory volume in one second): the volume of air expired in the first second of maximal expiration after a maximal inspiration. This is a measure of how quickly the lungs can be emptied.
2) FVC (forced vital capacity): It is the maximum volume of air that can be exhaled during a forced manoeuvre.
3) FEV1/FVC: FEV1 expressed as a percentage of the FVC, gives a clinically useful index of airflow limitation.
16
Fig 1: Normal flow volume loop in spirometry SPIROMETRY
Fig 2: Flow volume loop in Obstruction lung disease
17
Table 2: GOLD criteria for COPD severity [4]
GOLD stage
Severity Spirometry
I Mild FEV1/FVC <0.7 and FEV1≥80% predicted
II Moderate FEV1/FVC <0.7 and 50% ≥ FEV1 <80% predicted III Severe FEV1/FVC <0.7 and 30% ≤ FEV1 <50% predicted IV Very severe FEV1/FVC <0.7 and FEV1 <30% predicted
The most important disturbance of respiratory function in COPD is obstruction to forced expiratory airflow. During the last three decades lung function tests have evolved from tools for physiological study to clinical tools widely used in assessing respiratory states. In addition to their use in clinical case management, they have become a part of routine health examinations in respiratory, occupational and sports medicine and in public health screening.
MANAGEMENT[4,49-52]
The goals of COPD management include:
- Prevent disease progression - Relieve symptoms
- Improve exercise tolerance - Improve health status
- Prevent and treat complications - Prevent and treat exacerbations
18
- Reduce mortality
- Prevent or minimize side effects from treatment Fig 3: Assessment of COPD
In addition to spirometry, the following other tests should be undertaken for the assessment of a patient with moderate (stage II), severe (stage III) and very severe (stage IV) COPD.
Reduction of risk factors:
Smoking cessation: It is achieved by Counselling, Nicotine replacement products (like nicotine gum, inhaler, nasal spray, transdermal patch, sublingual tablet, or lozenge), and Pharmacological drugs like Varenicline.
Occupation exposures: emphasize primary prevention, which is achieved by elimination or reduction of exposures in the work place.
19
Secondary prevention achieved through surveillance and early detection is also important.
Indoor and Outdoor Air Pollution: Implement measures to reduce or avoid indoor air pollution from biomass fuel, used for cooking and heating in poorly ventilated areas. Advice patients to monitor public announcement of air quality and depending on the severity of their disease, avoid vigorous exercise outdoors or stay indoors altogether during pollution episodes.
Management of Stable COPD
Pharmacologic treatment can improve and prevent symptoms improve health status and improve exercise tolerance. It reduces the frequency and severity of exacerbations.
Bronchodilators: These medications are central to symptom management in COPD.
- Give as “needed” to relieve intermittent or worsening symptoms, and as a regular basis to prevent or reduce persistent symptoms.
- The choice between beta 2 agonists, anticholinergics, methylxanthines and combination therapy depends on the availability of medications and each patient’s individual response in terms of both symptom relief and side effects.
- Regular treatment with long acting bronchodilators is more effective and convenient than short acting bronchodilators, but more expensive.
20
- Combination of drugs with different mechanisms and duration of action may help to increase the degree of bronchodilation for with fewer side effects.
- Theophylline is effective in COPD, but due to its side effects, inhaled bronchodilators are more preferred when available.
- Regular nebulisation with bronchodilator therapy for a stable patient is not appropriate unless it has been shown to be better than conventional doses by metered dose inhaler.
Fig 4: Management of COPD
Global Strategy for Diagnosis, Management and Prevention of COPD
Manage Stable COPD: Pharmacologic Therapy
(Medications in each box are mentioned in alphabetical order, and therefore not necessarily in order of preference.)
Patient Recommended First choice
Alternative choice Other Possible Treatments
A
SAMA prn or SABA prn
LAMA or LABA
or SABA and SAMA
Theophylline
B
LAMA or LABA
LAMA and LABA SABA and/orSAMA Theophylline
C
ICS + LABA or LAMA
LAMA and LABA or LAMA and PDE4-inh. or
LABA and PDE4-inh.
SABA and/orSAMA Theophylline
D
ICS + LABA and/or LAMA
ICS + LABA and LAMA or ICS+LABA and PDE4-inh.or
LAMA and LABA or LAMA and PDE4-inh.
Carbocysteine N-acetylcysteine SABA and/orSAMA
Theophylline
21
Glucocorticoids: In Patients with FEV1< 50%, the appropriate treatment is regular treatment with inhaled glucocorcoids. Prolonged treatment with inhaled corticosteroids may relieve symptoms in this carefully selected group of patients but does not modify the long-term decline in FEV1. The relationship between dose response and long-term safety of inhaled glucocorticoids in COPD are not known.
Long term treatment with oral glucocorticoids is not recommended.
Vaccines: Influenza vaccines reduce 50% of serious illness and death in COPD patients. The vaccine has to be given once or twice every year. There is no evidence for recommending the general use of pneumococcal vaccine for COPD.
Antibiotics: Not recommended except for treatment of infections and exacerbations.
Mucolytic (Mucokinetic, Mucoregulator) agents: Patients with viscous sputum may benefit from mucolytics, but overall benefits are very small. Use is not recommended.
Antitussives: Regular use contraindicated in stable COPD.
Respiratory stimulants: Not recommended for regular use.
Non-pharmacological treatment: includes rehabilitation, oxygen therapy and surgical interventions.
22
Table: 3 Non – pharmacological management of COPD Patient
Group
Essential Recommended Depending on local guidelines
A
Smoking cessation
(can include pharmacologic
treatment)
Physical activity
Flu vaccination Pneumococcal vaccination
B, C, D
Smoking cessation
(can include pharmacologic
treatment) Pulmonary rehabilitation
Physical activity
Flu vaccination Pneumococcal vaccination
Rehabilitation programs should include at a minimum - Exercise training
- Nutrition counselling - Education
Oxygen therapy: In patients with chronic respiratory failure the long-term administration of oxygen for more than 15 hours per day, increases survival and has a beneficial impact on pulmonary arterial pressure, polycythemia (hematocrit > 55%), exercise capacity, lung mechanics and mental state.
The goal of long-term oxygen therapy is to increase the baseline PaO2 at rest to atleast 8.0 KPa (60 mmHg) at sea level, and/or produce SaO2 atleast 90%, which will preserve vital organ function by ensuring an adequate delivery of oxygen.
23
Initiate oxygen therapy for patients with stage IV (very severe COPD) with evidence of pulmonary hypertension, peripheral edema suggesting heart failure or polycythemia, if PaO2 is at or below 7.3 KPa (55 mmHg) or SaO2 is at or below 88%, with or without hypercapnea; or PaO2 is between 7.3 KPa (55 mmHg) and 8.0 KPa (60 mmHg) or SaO2 is 89%.
Surgical Treatment: Bullectomy and lung transplantation may be considered in carefully selected patients with stage IV i.e. very severe COPD. There is currently no sufficient evidence that would support the widespread use of lung volume reduction surgery (LVRS).
There is no evidence that mechanical ventilator has a role in the routine management of stable COPD.
Management of Exacerbations
COPD is often associated with exacerbation of symptoms. Many exacerbations are caused by infection of the tracheobronchial tree or an increase in air pollution. The cause for one- third of severe exacerbations cannot be identified.
Home care or hospital care for end-stage COPD patients:
In the presence of serious co morbidities, the risk of dying from an exacerbation of COPD is closely related to the development of respiratory acidosis, and the need for ventilatory support. Patient with severe underlying COPD often require hospitalization in any case.
24
Home Management
Bronchodilators: Increase dose and/or frequency of existing bronchodilator therapy. If not already used, add anticholinergics until symptoms improve.
Glucocorticosteroids: If baseline FEV1 < 50% predicted, add 40 mg oral prednisolone per day for ten days to the bronchodilator regimen. During exacerbations budesonide nebulisation is an alternative to oral glucocorticosteroids .
Antibiotics: When symptoms of breathlessness and cough are increased and sputum is purulent and increased in volume, provide antibiotic coverage of the major bacterial pathogens involved in exacerbations, taking into account local patterns of antibiotic sensitivity.
VITAMIN D: [53-73]
HISTORY OF VITAMIN D: [53-65]
Discovery of Vitamin D:
Sir Edward Mellanby, Great Britain had been very concerned with the extremely high incidence of rickets in Scotland. In fact, the disease became known as
‘the English Disease’.[54] Sir Mellanby was taken by the work of McCollum and found that rickets might be due to deficient in intake of proper nutrition. He very cleverly used oatmeal and fed that to dogs that he inadvertently kept away from sunlight. They developed rickets, which was identical to the human disease .[55]
Sir Mellanby[55] cured the disease by providing cod liver oil. He thought that it was vitamin A, which was responsible for the prevention of rickets. In Johns
25
Hopkins University, McCollum had been following this, and decided to test the hypothesis of vitamin A regarding healing of rickets. He bubbled oxygen through cod liver oil that destroyed vitamin A and found that this preparation was no longer able to prevent xerophthalmia, but it still retained the ability to cure rickets.[56]
McCollum et al.[56] correctly concluded that rickets is cured by a new vitamin, called vitamin D.
Healing of Rickets by UV Light In the meantime, Huldshinsky,[57] a physician in Vienna, and Chick et al.[58] in England found that children suffering from rickets could be cured by exposing them to UV light.
Hess and Unger[59] also noted that sunlight could cure rickets. This dichotomy attracted Professor Harry Steenbock at the University of Wisconsin who had been assigned the small animal experimental work. Steenbock in 1916 had been working with goats when he found that when they were kept in summer sun outdoors, they were in positive calcium balance but when kept indoors in the winter in the absence of sunlight, they went into negative calcium balance.[60]
Steenbock had then mentally made a connection between sunlight and calcium retention. With this background, Steenbock[61,62] began to irradiate rats, their food and the air in their cages with UV light. He found that irradiation of not only the rat but also their food could prevent or cure rickets. He concluded that an inactive lipid in the diet and skin could be converted by UV light into an active antirachitic Substance.[63]
26
Professor Steenbock[64] patented the process, and with this patent was able to attract industry to use this discovery to eliminate rickets as a major medical problem.
Hess and Weinstock[65] later discovered that irradiation could prevent rickets.
Vitamin D being an important fat soluble vitamin plays an essential role in regulation of calcium, magnesium and phosphate metabolism in our body. It helps in development and proper maintenance of bone.[66]
Vitamin D is synthesised in our body. Around 80% of the required vitamin D is directly synthesised by the skin on exposure to sunlight by the action of ultraviolet radiation on either 7-dehydrocholesterol or ergosterol. In addition both Vitamin D2 and D3 are present adequately in foodstuffs like oily fish, dairy products, eggs, and cereals. Nowadays, Vitamin D is available in a range of fortified food products and dietary supplements.
Vitamin D is produced in the skin by the exposure on exposure to the sunlight, and also absorbed from the foods containing vitamin D. 1, 25-dihydroxyvitamin D (1,25(OH)2D), is biologically active form, regulated calcium and phosphate metabolism.
Vitamin D deficiency will lead impaired formation of bone, which produces rickets in childrens and osteomalacia in adults.
Biochemistry and Physiology:[67]
Ergocalciferol (D2) and Cholecalciferol (D3) are the metabolites of vitamin D.
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VITAMIN D3:
Cholecalciferol is the naturally occurring, produced in the skin from 7- dehydrocholesterol on exposure to the ultraviolet B rays of the sunlight.[68, 69] Climate, latitude, age, sunscreen use also influence its production.
VITAMIN D2:
It is produced by the irradiation of ergosterol produced by yeast. Vitamin D3 differs from vitamin D2 by a double bond between C-22 and C-23, methyl group on C-24.
SOURCES:
Fish liver oil, fatty fish, egg yolk and liver are food sources contain vitamin D.
RECOMMENDED DAILY ALLOWANCE:
RDA is 400IU (10µg)
METABOLISM AND REGULATION:[70,69]
Vitamin D2 and vitamin D3 are metabolized to 25- hydroxyvitamin D [25(OH)D] in the liver by vitamin D 25-hydroxylase, a cytochrome P450 enzyme.[70,69]
28
Fig 5: Synthesis of vitamin D3
Fig 6:Generation of vitamin D3 in Human body
29
Fig 7: Synthesis of vitamin D from sunlight
30
Vitamin D binding protein (DBP):[68,71,72]
In circulation, vitamin D and its forms are bind to vitamin D binding protein (DBP), a high affinity transport protein called as group –specific components of serum or Gc- globulin. [68, 71, 72]
D-binding protein belongs to the albumin and α-fetoprotein. It contains 458 aminoacids residues, molecular mass 51,335 Da. DBP is synthesized by the liver at about 400mg/L.
only 5% is occupies by vitamin D.
DBP binds particularly the 25- hydroxylated metabolites, 25(OH)D, 24,25(OH)2D, and 1,25(OH)2D.[68,72] Only 0.03% of 25(OH)D and 0.4% of 1,25(OH)2D are freely available in plasma.
The concentration of DBP is increased in pregnancy and in patients on estrogen therapy, decreased in nephritic syndrome patients.
Biological action of 1, 25- Dihydroxyvitamin D: [68, 71, 70]
It helps to maintain calcium and phosphate metabolism by this action on intestine, kidney and parathyroid glands. In small intestine it stimulates calcium absorption initially in the duodenum and phosphate absorption by jejunum and ileum.[68, 71, 70]
The calcium in the food is absorbed by three events:
1) Calcium entry in brush border cytoplasm, mediated by epithelial Ca2+
transporter or channel
2) Diffusion of calcium within the cell by calbindin-D9k, a cytosolic calcium binding protein.
31
3) Exit of calcium across its basolateral membrane by action on CaATPase.
Approximately 90% of CaT1 synthesis and complete calbindin D synthesis is vitamin D dependent.[73, 74]
Calcium homeostasis: When serum calcium is low, PTH is stimulated, resulting in increased calcium release from bone and decreased renal calcium excretion. PTH also stimulates increased production of calcitriol, which acts to increase absorption of calcium from intestine.
Fig 8: Absorption and regulation of Vitamin D3
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ABNORMAL CIRCULATING CONCENTRATIONS OF 25-HYDROXY VITAMIN:[67]
Decreased 25(OH) D:
- Less exposure to sunlight - Insufficient dietary vitamin D - Vitamin D supplementation - Severe hepatocellular disease
- Increased catabolism (drugs e.g., anticonvulsants) - Increased loss(nephritic syndrome)
- In hepatic insufficiency, osteodystrophy is common due to an inability of the liver to transform vitamin D into its metabolically active form.
DEFICIENCY MANIFESTATIONS:
Rickets in childrens.
Osteomalacia in adults Clinical features:
- Bow legs , knock knee - Ricketic rosary
- Osteoporosis.
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Fig 9: BOW LEGS:
Fig 10: RICKETIC ROSARY
Fig 11: NORMAL BONE AND OSTEOPOROTIC CHANGES IN BONE
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Increased 25(OH) D:
Vitamin D intoxication
ROLE OF PARATHORMONE IN VITAMIN D :
TYPES OF GLAND
- Endocrine Glands are the glands which contain no duct and release their secretions into the intercellular fluid or directly into the blood. The compilation of endocrine glands makes up the endocrine system.
- The important endocrine glands are the pituitary (anterior and posterior lobes), thyroid, parathyroid, pancreas, adrenal (cortex and medulla), and gonads.
HORMONES AND TYPES
The endocrine system secretes hormones that are important in maintaining regulation of reproduction, homeostasis and development. A hormone is a chemical messenger secreted by a cell that effects specific change in the cellular activity of the target cells. But exocrine glands which secrete substances such as milk, saliva, stomach acid and digestive enzymes. Endocrine glands do not secrete substances into ducts (tubes). Instead, they secrete their hormones directly into the surrounding extra cellular space. The hormones then enter into the nearby capillaries and then they are transported throughout the body in the blood.
35
PARATHYROID GLAND: [67]
The parathyroid gland plays an major role in regulation of the metabolism of 25-OHD3, primarily in controlling the conversion to 1,25-(OH)2D3, the active form of vitamin, by increasing intestinal calcium transport and in increasing mobilization of calcium from bone. The results suggest that parathyroid hormone may serve as a a tropic hormone that ultimately stimulates 1, 25-(OH)2D3 synthesis. Parathyroid hormone must be considered essential for the functioning of physiologic amounts of vitamin D. [67]
The vitamin D must first be activated or "turned on" by parathyroid hormone (PTH). Once activated, vitamin D acts to greatly increase the amount of calcium that the intestines can absorb from food, sometimes by as much as two to four times. The body can either make its own vitamin D using a process that requires sunlight or obtain vitamin D directly from the diet. [67]
Chemiluminescence immunoassay:[67]
It is a chemical reaction which causes, the emission of light when the electrons return from an excited energy level to a lower energy level. Also involves in oxidation of an organic compound such as luminol, isoluminol etc., It is ultrasensitive.
COPD VS VITAMIN D[75-78,90-96]
Various study done by Herr C et al, Banerjee A et al, Janssens W et al, suggested that the precise role of vitamin D in the pathogenesis of COPD is unclear however studies have described that vitamin D can alter the activity of various immune cells, regulate airway smooth muscle and inhibit inflammatory responses.[75,76,77]
36
Islam S et al. found out that 37% of the COPD patients vitamin D deficiency (<20ng/ml) and 28.75% of COPD patients had vitamin D insufficiency (20-29ng/ml) and it was found to be significantly lower than the health controls.[78] The study also revealed that the vitamin D deficiency was significantly associated with exacerbation of COPD.
Mattila T et al. in their cohort study revealed that obstruction and vitamin D independently each other predict the mortality among COPD patients.[79]
Another study by Mendy A et al “Blood biomarkers as predictors of long – term mortality in COPD” showed that high CRP, Eosinophil count <2%, hypoalbuminemia and hypovitaminosis are associated with poor prognosis in COPD patients.[80]
In a randomised control trial by Jolliffe DA et al. studied and exhibited that the supplementation of Vitamin D reduced the exacerbations among COPD patients with baseline 25-hydroxyvitamin D <25nmol/L.[81]
A Poland based study by Gawron G et al revealed that there are very frequent hypovitaminosis D from COPD patients in respiratory failure and also even among healthy persons in autumn to winter season.[82]
A Japanese based study on “Secondary osteoporosis in Chronic obstructive pulmonary disease: COPD” suggested that supplementation of Vitamin D supplements and antiosteoporotic measures would prevent COPD patients suffering from fracture.[83]
Kim SH et al in their study on “Sarcopenia associated with chronic obstructive pulmonary disease” revealed that chronic inflammation, oxidative stress, inactivity,
37
hormone instability & inactivity, hypoproteinemia, hypoxemia, hypovitaminosis D were the factors associated with decrease muscle power and strength leading to Sarcopenia.[84]
Franco CL et al in their study on revealed that Low blood levels of 25- hydroxyvitamin D have been associated with a higher risk of respiratory infections in general populations & higher risk of exacerbations of lung disease in people with asthma and COPD. [85]
Batlle JD et al in their study on “Dietary habits of firstly admitted Spanish COPD patients” revealed that the dietary intake of vitamin D is reduced in COPD patients particularly in elderly.[86]
Ito I et al studied “Risk and severity of COPD is associated with the group- specific component of serum globulin 1F allele” and revealed that patients with certain gene variants of the vitamin D transport protein shows significant correlation between serum levels of vitamin D and severity of COPD. [87]
Vitamin D supplementation in COPD:
Martineau et.al in his study found that patients with COPD often have vitamin D deficiency. It is associated with increased susceptibility to upper respiratory tract infections, which precipitates exacerbations in COPD patients. Multicentre trials of vitamin D supplementation in COPD patients for preventing upper respiratory tract infections and exacerbations are lacking. He concluded that Vitamin D3 supplementation protected against moderate or severe exacerbation in patients with baseline vitamin D less than 50 nmol/L (20ng/mL). But the respiratory tract infection is not controlled.[88]
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Bellocchia M et al in his study found that 25-Hydroxyvitamin D deficiency is frequent in COPD patients. It favours exacerbations and hospitalization in COPD patients. After vitamin D supplementation there is a significant decrease in AECOPD and HCOPD. [89]
Ferrari R, Caram LMO et al found that vitamin D supplementation plays a role in preventing exacerbations and hypovitaminosis D in COPD patients.[90]
39
MATERIALS AND METHODS
A case control study was carried out during the period of February 2018 to June 2019 among all types were used for the investigation. Chronic obstructive pulmonary disease patients and healthy volunteers with age group of more than 30 to 75 years were selected for the study. The total numbers of samples for the study were 50 cases and 50 control attending the outpatient &
inpatient Department of Pulmonary medicine and General medicine, Karpaga Vinayaga Institute of Medical Sciences. The controls were the persons attending regular health checkup who have no known significant medical illness which can affect the outcome of the study.
This study was approved by the institutional ethical committee. Age and Body mass index was quantified by bioelectrical impedance analysis.
Bioelectrical impedance analysis:
It is a method for measuring body composition based on the rate which an electrical current travels through the body. Body fat causes greater resistance than fat-free mass, which slows at which the current travels.
Vitamin D3, Plasma glucose, renal function test along with a written consent was taken from every patient (Cases & Controls). Blood samples from cases as well as control group were obtained to determine the following investigations.
Vitamin D3 was measured by Chemiluminescence Immunoassay (CLIA) method, using Maglumi 1000 kit.
40
Biological Reference Interval:
Vitamin D3: 30-100ng/mL.
Plasma glucose fasting (70-110 mg/dl) and post prandial (upto 130mg/dL)
Renal function test:
Blood urea (15- 40 mg/dl), Serum creatinine (Male: 0.9-1.4 mg/dl;
Female 0.8-1.2 mg/dl)
Done by CLIA, GOD/POD method, enzymatic method & Jaffe’s method respectively.
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ASSESMENT PARAMETER:
1. Age: (30-75years)
2. Gender (males and females)
3. Personal History: Smoking, Exposure to fuel woods, occupational exposure, chemical exposure.
4. Body mass index
5. Known case of COPD – Number of Exacerbations, Grades of COPD and treatment.
EXCLUSION CRITERIA:
1. Patients less than 30 years of age
2. Patients taking vitamin D supplementation
3. Patients suffering from diseases which affects Vitamin D and calcium metabolism like Renal dysfunction, Osteomalacia, Malignancy, Thyroid disorders, Parathyroid dysfunction, , Inflammatory bowel diseases, History of small bowel resection, Cholestatic liver disease, Pancreatitis, Cystic fibrosis, Bronchiectasis, Granulomatous disorder.
4. Patient on treatment with drugs like Phenytoin, Phenobarbital, Carbamazepine, Isoniazid, Rifampin, Tenofovir, Efavirenz
5. Patients not willing to take part in the study
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Patients who are consenting to participate in the study and fulfilling inclusion
& exclusion criteria will be subjected to complete history taking, physical examination, plasma glucose, blood urea, serum creatinine, serum electrolytes &
serum Vitamin D level estimation. Vitamin D levels are measured using Chemiluminescence immunoassay (CLIA). Appropriate statistical methods will be used to analyse the study.
HISTORY
History was elicited according to Pro forma enclosed in the annexure. In a known COPD patient importance was given to their personal history, occupational history, and treatment history. History was the first level for the selection process of
individuals to include them in the study.
GENERAL AND SYSTEMATIC EXAMINATION:
General examination including anthropometric measurements and systematic examination was done according to the proforma attached in the annexure.
BODY MASS INDEX CALCULATION:
Body mass index (BMI) was calculated by dividing weight (Kg) by height squared (m2). They were classified by WHO as follows:
43
WHO Classification for BMI
NUTRITIONAL STATUS BMI (kg/m2)
UNDERWEIGHT <18.50
NORMAL 18.50 – 24.9
PRE OBESE 25-29.99
OBESITY CLASS I 30.0 – 34.9 OBESITY CLASS II 35.0 – 39.9
OBESITY CLASS III ABOVE 40
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ESTIMATION OF 25-OH VITAMIN D (CLIA): [91]
METHOD:
Quantitative determination of 25-OH Vitamin D in human serum using SNIBE fully auto analyzer (Maglumi1000).
PRINCIPLE:
Competitive immune luminometric assay:
Using a purified 25-OH Vitamin D antigen to label ABEI (N- (aminobutyl)-N- (ethylisoluminol)), and use 25-OHVitamin D monoclonal antibody to label FITC (Fluorescein isothiocyanate). Sample, Calibrator, or Control, Displacing reagent, FITC Label and magnetic microbeads coated with anti-FITC are mixed thoroughly and incubated at 37 ℃, forming antibody-antigen complexes. After sediment in a magnetic field, decant the supernatant, then cycle washing for one time. Then add ABEI Label, incubation and washing for the 2nd time, sample antigen and ABEI labeled antigen compete to combine with FITC labeled monoclonal antibody, forming antibody-antigen complexes. Subsequently, the starter reagents are added and a flash chemiluminescent reaction is initiated. The light signal is measured by a photomultiplier as RLU (Relative light unit) within 3 seconds and is proportional to the concentration of 25-OH Vitamin D present in controls or samples.
SAMPLE:
Unhemolysed serum
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REAGENTS:
REAGENT INTEGRAL FOR 100 DETERMINATIONS Nano magnetic micro beads 2.5mL
Calibrator low 3 mL
Calibrator high 3 mL
Displacing reagent acidic buffer 6.5 mL ABEI label:25-OH Vitamin D antigen
labeled ABEI
22.5 mL
FITC label: 25-OH Vitamin D monoclonal antibody labeled FITC
12.5 mL
All reagents are provided ready to use
REAGENT PREPARATION AND STABILITY:
All reagents are provided ready to use
Sealed: Stored at 2-8℃ until the expiry date.
Opened: Stable for 4 weeks.
PROCEDURE:
100µL Sample, calibrator or control
+100μL +50μL +20Ml
FITC Label
Displacing reagent
Nano-magnetic microbeads
20 min Incubation
400µL each time Cycle washing
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+200 µL ABEI Label
10 min Incubation
400µL each time Cycle washing
3 Sec Measurement
BIOLOGICAL REFERENCE INTERVAL:
Adults: 30- 100ng/mL
ESTIMATION OF GLUCOSE: [67]
METHOD:
GOD- POD (Glucose oxidase /peroxidase) Method (Trinder 1969) [67]
PRINCIPLE:
Glucose is oxidised to gluconic acid and hydrogen peroxide in the presence of glucose oxidase. Hydrogen peroxide further reacts with phenol and 4- amino antipyrine by the catalytic action of peroxidases and form a red quinineamine dye. Intensity of colour formed is directly proportional to the concentration of glucose present in the patient sample.
REACTIONS
Glucose + O2 + H2O Glucose oxidase Gluconic acid + H2O2
H2O2 + 4-amino antipyrine + phenol peroxidase Red quinine aimine dye + H2O
SAMPLES PREPARATION:
Blood is collected in a tube containing sodium fluoride and potassium oxalate, mixed
47
well and centrifuged for 10 mins at 3000rpm (Revolutions per minute). The separated plasma is used for analysis.
REAGENTS:
Standard Glucose: 100 mg/dL Glucose reagent enzyme.
PROCEDURE:
Sample Size: 3 µL
Reagent Volume: 300 µL
Wavelength: Measured primarily at 505 nm and secondarily at 670 nm.
Type of Measurement: Bichromatic endpoint.
BIOLOGICAL REFERENCE INTERVAL:
Fasting plasma Glucose: 70-110mg/dL
Post Prandial plasma Glucose: Up to 130mg/dL.
ESTIMATION OF BLOOD UREA: [92]
METHOD:
Quantitative estimation of Blood Urea in Biosystems BA 200 Clinical chemistry analyser by Urease / GLDH (Glutamate dehydogenase) method [92]
PRINCIPLE:
Urea in the sample consumes, by means of the coupled reactions described below, NADH that can be measured by spectrophotometry.
Urease
Urea + H2O 2NH4+ + CO2
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GDH
NH4+ + NADH + H+ + 2 – oxoglutarate Glutamate + NAD+ SAMPLE:
Unhemolysed serum.
REAGENTS:
Reagent 1 - Tris 100 mmol/L 2-oxoglutarate 5.6 mmol/L Urease > 140 U/mL Ethyleneglycol 220 g/L Reagent 2 NADH 1.5 mmol/L Sodium azide 9.5 g/L Standard Urea 50 mg/dL PROCEDURE:
Sample Size: 3 µL
Reagent 1 Volume: 240 µL Reagent 2 Volume: 60 µL Test Temperature: 37°C Wavelength: 340 nm
Type of Measurement: monochromatic endpoint BIOLOGICAL REFERENCE INTERVAL:
15-39 mg/dL
49
ESTIMATION OF SERUM CREATININE: [92]
METHOD:
Quantitative estimation of Serum Creatinine in Biosystems BA 200 Clinical chemistry analyser by Jaffe’s method. [92]
PRINCIPLE:
Creatinine in the sample reacts with picrate in alkaline medium forming a coloured complex (Jaffe’s method). The complex formation rate is measured in a short period to avoid interferences. Serum and plasma samples contain proteins that react in a non specific way; nevertheless, the results can be corrected subtracting a fixed value.
The use of this correction is known as the Jaffe’s method compensated.
Creatinine + Picric acid Alkaline medium Orange Coloured Complex SAMPLE:
Unhemolysed serum REAGENTS & STORAGE:
Reagent 1: Creatinine Buffer reagent Reagent 2: Creatinine Picrate reagent Reagent 3: Creatinine Standard 2 mg/dL
Working reagents and Creatinine standard with value 2mg/dl were ready for the assay and stable till the expiry date when stored at 2- 8ºC
PROCEDURE:
Sample Size: 20 µL Reagent 1 :100 µL
50
Reagent 2 : 100 µL Test Temperature: 37°C Wavelength: 505 nm
Type of Measurement: monochromatic endpoint.
BIOLOGICAL REFERENCE INTERVAL:
Men: 0.9 to 1.3 mg/dL Women: 0.6 to 1.1 mg/dL
ESTIMATION OF HEMOGLOBIN AND TOTAL LEUCOCYTE COUNT: [93]
AIM:
Quantitative enumeration of different formed elements of blood and Hemoglobin and Total WBC count in whole blood by automated cell counter The Principle of XT 2000i performs analysis of WBC’s with an optical detector block based on the flow cytometry method. Hemoglobin bared on the SLS hemoglobin detection method. Reference values vary across the life cycle and between sexes and differ in different age groups and in various disease conditions.[93]
PRINCIPLE:
Hemoglobin: During an HGB analysis the amount of hemoglobin in the blood is measured. The procedure for analyzing HGB is explained here.
Blood is aspirated from the manual aspiration pipette to the sample rotor valve. 3.0µl of blood diluted with 0.9970 ml of cell pack and sent to flow
51
cell at the same time 0.5 ml of salfolyser added to hemolyse the red cells to make 1:500 diluted and the shemoglobin is converted into SLS hemoglobin.
The light (555nm) emitted from the light emitting diode passes through the lens and into the sample in the HgB cell. The concentration of SLS hemoglobin is measured as light absorbance, and is calculated by comparison with the absorbance of the diluent measured before the sample was added
WBC: After a predetermined volume of blood is aspirated and diluted by a certain amount of reagent is infected in to the flow cell. Forward slathered light and lateral fluorescent light are detected (via) flow cytometry method utilizing a semiconductor laser and two dimensional scatter grams and drawn in a 4 diff. scatter gram. The X axis represents the intensity of the lateral scattered light, and the Y-axis the intensity of the lateral fluorescent light in a WBC/BASO scatter gram. The X-axis represents the intensity of the lateral scattered light and the Y-axis the intensity of the forward scattered light. A 4 diff. analysis scatter gram displays the classified groups of red blood cells ghosts, lymphocytes, monocytes, eosinophils, neutrophils and basophils.
The HPC function of the HORIBA PENTRA 60S ANALYSER capable of monitor the HPC (Hematopoietic Progenitor Cell) mobilized to the peripheral blood. The HPC is the parameter for research it is not be used as reporting data of results. When analyzing samples with HPC analysis mode,
